The rapid appearance of homostyly in a cultivated distylous population of Primula forbesii

Abstract Evolutionary breakdown from rigorous outbreeding to self‐fertilization frequently occurs in angiosperms. Since the pollinators are not necessary, self‐compatible populations often reduce investment in floral display characteristics and pollination reward. Primula forbesii is a biennial herb with distribution restricted to southwest China; it was initially a self‐incompatible distylous species, but after 20 years of artificial domestication, homostyly appeared. This change in style provides an ideal material to explore the time required for plant mating systems to adapt to new environmental changes and test whether flower attraction has reduced following transitions to selfing. We did a survey in wild populations of P. forbesii where its seeds were originally collected 20 years ago and recorded the floral morph frequencies and morphologies. The floral morphologies, self‐incompatibility, floral scent, and pollinator visitation between distyly and homostyly were compared in greenhouse. Floral morph frequencies of wild populations did not change, while the cultivated population was inclined to L‐morph and produced homostyly. Evidence from stigma papillae and pollen size supports the hypothesis that the homostyly possibly originated from mutations of large effect genes in distylous linkage region. Transitions to self‐compatible homostyly are accompanied by smaller corolla size, lower amounts of terpenoids, especially linalool and higher amounts of fatty acid derivatives. The main pollinators in the greenhouse were short‐tongued Apis cerana. However, homostyly had reduced visiting frequency. The mating system of P. forbesii changed rapidly in just about 20 years of domestication, and our findings confirm the hypothesis that the transition to selfing is accompanied by decreased flower attraction.

In numerous families, the shift of mating system will affect the biodiversity, the response to selection, the evolution of floral signals and rewards, and the population structure (Charlesworth & Charlesworth, 1979;de Vos et al., 2014;Sicard & Lenhard, 2011;Yuan et al., 2017;Zeng et al., 2022). This transition has a profound ecological, evolutionary, and genetic influence on plant populations because it leads to reproductive isolation and subsequent speciation (Wright et al., 2013), which are interesting to biologists since Darwin's seminal research (Darwin, 1877;Yuan et al., 2019;Zhang et al., 2021;Zhou et al., 2017). Of special value for investigations of mating system changes are lineages that include outcrossing and self-pollination, which provide a valuable opportunity to determine the selection forces and evolutionary consequences of selfing transitions.
A paradigmatic model for studies of shift from outcrossing to selfing is the evolutionary breakdown of the heterostyly to homostyly. Heterostyly is a genetically controlled floral polymorphism and includes two (distyly) or three (tristyly) floral morphs that differ reciprocally in stigma and anther position (Barrett, 1992;Darwin, 1877;Lloyd & Webb, 1992) and has arisen independently in at least 28 angiosperm families and 199 genera (Barrett, 2019). The major type of heterostyly is distyly, in which the flowers of one morph have a high stigma and low anthers (long style morph, L-morph), while the other morph has a low stigma and high anthers (short style morph, S-morph; Eckert . Heterostylous plants possess heteromorphic self-incompatibility, which prevents self-and intra-morph pollinations and polymorphism in ancillary traits, such as dimorphism of pollen grains and stigma papillae (Barrett & Cruzan, 1994).
Based on the genetic studies of heterostyly, homostyly can arise from rare crossovers and/or mutations of the S-locus linkage genes (Barrett & Shore, 2008;Charlesworth & Charlesworth, 1979;Ernst, 1955;Li et al., 2016;Yuan et al., 2019), or unlinked modifier genes that have small effects and are not linked with S-locus (Ganders, 1979;Mather & de Winton, 1941). The homostyly formed by modifiers is characterized by many phenotypic variations in the positions of stigmas and anthers (Brys & Jacquemyn, 2015). The patterns of phenotypic variation in heterostylous populations have important implications for the origin pathway of the homostyly (Yuan et al., 2017), although the accurate conclusion depends on further genetic analysis.
Consistent with the shift from outcrossing to autonomous self-fertilization in monomorphic species, the transition to homostyly in heterostylous groups is accompanied by remarkable floral morphology and metabolite changes. In these autogamous plants, the flowers have a reduced corolla, nectar and pollen yield, low floral scent emission, and a lost herkogamy (stigma-anther separation) (de Vos et al., 2014;Sicard & Lenhard, 2011;Wu et al., 2017;Zeng et al., 2022;Zhong et al., 2019). The floral scent is a key ecological adaptation signal for interacting plants and pollinators (Farré-Armengol et al., 2013;Majetic et al., 2009). More than 1700 floral scent components exist, including terpenoids, benzenoids, and fatty acid derivatives (Knudsen et al., 2006). These promote outcrossing and propagation of flowers by luring insect pollinators and will reduce emission and/or change chemical composition after a transition from outcrossing to selfing (Doubleday et al., 2013;Majetic et al., 2019;Petrén et al., 2021;Sas et al., 2016). Although considerable studies have been done on the transition of mating system in monomorphic groups, few studies have focused on the changes of floral scent in the shift from heterostyly to homostyly (Zeng et al., 2022).
Primula (Primulaceae) is a well-known distylous genus, with a lot of concerns for more than a century since Darwin's pioneering research (Darwin, 1877). The majority of the 430 species are distylous (95%), while the remaining are homostylous (de Vos et al., 2014;Mast et al., 2006;Richards, 2003). Phylogenetic analysis of Primula indicates a single independent origin of distyly and multiple disruptions to homostyles in the genus (Mast et al., 2006). Intraspecific observations of some Primula populations further indicate that homostyly evolved from distylous plants (Brys & Jacquemyn, 2015;Zhou et al., 2017). Primula forbesii Franch. (Figure 1a) is a less-known distylous taxon with a strong self-incompatibility and a pleasant fragrance (Chen & Hu, 1990;Huu et al., 2022). However, preliminary observations indicate that after about 20 years of greenhouse cultivation, the distylous population of P. forbesii now varies in floral morphs, producing homostylous plants with long styles and high stamens (Figure 1b-d).
Although the appearance of homostylous plants in distyled populations is common, it has been rarely reported that the mating system transition can occur rapidly in just about 20 years. We, therefore, firstly returned to wild populations where the P. forbesii seeds were collected originally and documented the floral morph frequencies and floral morphologies to determine whether the homostyly also appeared. Furthermore, we determined the effects of changes from distyly to homostyly on floral morphologies, self-incompatibility, floral scent, and pollinator visitation to test whether the transition to selfing reduces investment in character-
In the spring of 2000, the full-grown P. forbesii plants were  collected in the spring of 2019, were sown in the seedling tray in July 2020 in the greenhouse at Sichuan Agricultural University. After 1 month, more than 3000 seedlings were transplanted into 10 × 15 cm plastic pots containing peat and soil and watered every 3 days until flowering.

| Floralmorphfrequencyandmorphological characteristics
We conducted field investigations in the spring of 2020 and 2021 recorded floral morphs in 316 HLT and 389 XS P. forbesii flowering individuals. For the cultivated population CD, we randomly set up 10 quadrats of 10 × 10 pots, each at an interval of 1-2 m, and recorded floral morphs. To sample randomly, we did not replace plants that had not bloomed or died in the quadrats. Therefore, the effective sample size of population CD is 936 plants. We used χ 2 -tests using SPSS version 23.0 (IBM Corp., Armonk, NY, USA) to determine whether the frequencies of L-and S-morph in cultivated and wild populations had deviated from the expected balanced ratio of 1:1.
For the floral morphological traits, the newly opened flowers, including 40 of each L-, S-, and H-morphs from HLT, XS, and CD populations, were collected and slit longitudinally. The stigma and anther height, herkogamy (stigma-anther separation), length and width of the corolla tube, and corolla diameter were measured using a digital calliper with an accuracy of 0.01 mm (Brys & Jacquemyn, 2015). About 10-15 buds were collected from 10 plants of each floral morph of three populations to count pollen grains and ovules. The anthers were separated and dried in an oven at 40°C for 12 h, then mixed with distilled water in a 2 ml calibrated tube. The suspension was shaken for 30s, and three independent samples of 1 μl were separately used for slide preparation. The pollen grains were observed and photographed under an optical microscope and counted using ImageJ software . The ovaries were carefully dissected, and ovules counted under a stereomicroscope . We randomly collected 10 flowers from each floral morph to measure nectar. The nectar was collected using 7 μl micro-capillary tubes at 8:00-11:00 am after flowering. A portable refractometer (Brix 0%-80%; CJM-32, China) was then used to measure the total sugar content in each sample with seven replicates.
The shape, size, and exine sculpture of the pollen grains among different populations and floral morphs were determined in anthers collected during early anthesis from five plants. Dissected anthers were dried in an oven at 40°C for 12 h, followed by the scattering of pollen grains on copper stubs with conductive tapes and then observed under a scanning electron microscope (SU3500; Hitachi High-Tech, Ibaraki, Japan) with each sample replicated three times.
Pollen polar-and equatorial axis were measured using ImageJ. Three styles from three plants were collected to determine the variation of stigma papillae cells in L-, S-, and H-morph plants. The styles were placed in glutaraldehyde and osmium tetroxide and then dehydrated for 15 min with ethanol in a series beginning with 30%, followed by 50%, 60%, 70%, 80%, 90%, 95%, and 100%; (Massinga et al., 2005).
The characteristics of stigma were observed using a scanning electron microscope (EM CPD300; Leica; Germany) after critical point drying. We used a one-way analysis of variance (ANOVA) to test the significant differences in floral morphological and ancillary traits between cultivated and wild populations and a t-test to determine the considerable variations between homostyly and distyly in SPSS version 23.0.

| Pollenviabilityandlongevity
To compare pollen viability and longevity in different flower morphs, pollen grains from five plants of each floral morph in the cultivated population were collected on the 0, 2, 4, 6, 10, and 14th day after anthesis, respectively. The pollen was cultured in a medium containing 15% sucrose, 2 × 10 −3 M H 3 BO 3 and 2 × 10 −3 M Ca (NO 3 ) 2 , pH 5.6, in 25°C, and 90% humidity for 4h. The pollen grain was considered germinated when the pollen tube exceeded its diameter. More than 100 pollen grains were counted in every five repetitions to confirm the pollen germination rate in vitro. The significant differences in pollen viability among three floral morphs at different time intervals were determined based on a generalized linear model (GLM).

| Variationofself-incompatibility
We conducted artificial pollination in a glasshouse to confirm the pollen tube growth rate and self-incompatibility intensity of L-, S-, and H-morphs in the cultivated population of P. forbesii. The treatments included: (1) intermorph, intramorph, and selfing pollinations in the L-and S-morphs; and (2) intramorph and selfing pollinations in the H-morphs. To determine the pollen tube growth rate, each pollination type included 35 flowers from at least seven plants with five flowers fixed in 50% FAA for 12h per time interval (4, 8, 12, 24, 48, 96, and 144 h). After washing three times with distilled water, the styles were cleared in 8 mol L −1 NaOH solution at 25°C for 20 min, then rinsed three times again with distilled water before dying with 0.1% aniline blue for 25 min (Lu et al., 2018). More than 10 pollen tubes of each flower were then measured in transparent pistil tissue by fluorescence microscope (BX53-DP80; Olympus).
To elucidate the self-incompatibility intensity of L-, H-, and Smorph, 12-17 plants with about 150-250 flowers were artificially pollinated for each pollination type, using flowers from the second and third day after anthesis. Pollination plants were caged before and after artificial pollinations to exclude insect visitors from causing outcrossing, while inter-and intra-morph pollination flowers were emasculated before anthesis. For outcrossing pollinations, pollen was collected from more than three flowering plants and blended before deposition. Pollination experiments were carried out in February 2021 and the fruits matured in late March, after which fruit set and seed production per fruit were recorded. The GLM was used to compare fruit set and seed production per fruit among treatments for each floral morph in SPSS version 23.0.

| Analysisoffloralscentcompounds
Newly opened flowers from L-, S-, and H-morph of cultivated population of P. forbesii were collected between 7:00 am and 9:00 am during the blooming stage. We randomly took 0.6 g (approximately 15) flower samples from at least 10 plants of each flower morph with five replicates each. Flowers were introduced into 20 ml sterilized clear glass vials and analyzed within 10 min.
The headspace solid-phase microextraction (HS-SPME) and chromatography analyses were performed on a gas chromatographymass spectrometry (GS-MS) (TQ8050, Shimadzu, Japan) with an automatic sample manager system. Before headspace collection, the SPME (1.1 mm, CARBON-WR/PDMS) fiber head was thermally conditioned for 6 min at 250°C. Flowers were incubated for 5 min at 50°C before transferring the SPME fiber to the headspace of the vial for 30 min at 50°C to absorb floral scent compounds. Afterward, the fiber was moved to the injection port for thermally desorbing for 2 min at 250°C.
The chromatographic assay was performed on an InertCap Pure-WAX column (0.25 mm × 30 m × 0.25 μm). The carrier gas was highpurity helium, and the split mode was applied at a split ratio of 50:1.
The column flow rate was 1.43 ml min −1 , the linear velocity was 43.3 cm s −1 , and the purge flow rate was 3 ml min −1 . In the GC-MS analyses, a temperature program was set as follows: 50°C held for 5 min with a speed of 10°C per min up to 250°C for 2 min. The total operation time was 27.0 min. Mass spectrometry was done using the triple-quadrupole mass spectrometer (QQQ-MS/MS). The program was set as follows: the ionization source was electron ionization (EI), the collision gas was high-purity argon, and the source temperature Compounds were determined by comparing the collected fragments with those stored in the NIST libraries. Furthermore, the retention indices for each floral scent compound were obtained by n-alkane standards and compared with those issued in previous reports. We calculated the relative amounts of each volatile compound by dividing the single peak area by the sum of all peak areas and multiplying by 100%. The floral scent intensity was estimated using the total peak area of the floral sam-ple′s chromatogram.
Because the floral volatile data were not normally distributed, a nonparametric U-test was used to compare the relative amounts of each volatile compound between homostylous and distylous plants.
To determine the overall floral scent differences among the three flower morphs, nonmetric multidimensional scaling (NMDS) was carried out using Bray-Curtis similarity indices with 1000 permutations. In addition, a permutational multivariate analysis of variance (PERMANOVA) was also carried out using Bray-Curtis similarity indices with 1000 permutations to examine significant differences in total floral scent profile among the investigated three morphs (Okamoto & Su, 2021).

| Insectvisitorstoflowers
To demonstrate whether different morphs affect floral visitation under the same conditions, we established three P. forbesii forag- The experiments were conducted daily for 1 week, from 10:00 am to 15:00 pm. We recorded all visiting insects and counted the visiting number for each floral morph in each bout. Visiting insects were photographed, caught, and returned to the specimen room for identification. We calculated visitation frequency in each floral morph by dividing the number of visited flowers by the total amount of flowers in each array. We also compared visitation frequencies of morphs of P. forbesii using one-way ANOVA in SPSS 23.0.

| Floralmorphologyandancillarytraits
Flowers of plants growing in a cultivated environment possessed wider and longer floral tubes, larger floral diameters, and higher nectar yield and sugar content than flowers from wild populations (Table 2). However, the herkogamy, pollen-ovule ratio (P/O), and the polar and equatorial ratio of pollen (P/E) of flowers in cultivated populations reduced significantly relative to wild populations (p < .05; Table 2; Appendix S1). The pollen grains of plants grown in cultivated were spherical and spindle-shaped in wild populations (Figure 1e-g; Appendix S2). In the three populations, the height of stigma and anthers of L-and S-morph individuals displayed a discrete dimorphism, while the homostylous flowers in the cultivated population showed a unimodal distribution (Figure 2).
In the cultivated population, the homostyles possessed highlevel stigma (5.18 ± 0.04 mm) and anthers (5.01 ± 0.03 mm) but almost completely lost herkogamy (0.19 ± 0.02 mm) in comparison with L-(1.88 ± 0.05 mm) and S-morphs (2.37 ± 0.07 mm; p < .01; Figure 1b-d; Table 2). As expected, the homostyles of P. forbesii possessed pollen size close to S-morphs and stigma papillae near to L-morphs (Figure 1e-j). The corolla tube length of homostyles was significantly larger than L-morphs and smaller than S-morphs (p < .01); however, the corolla tube width and corolla diameter were significantly smaller than both the L-morphs and S-morphs (p < .01).
Furthermore, the pollen count of homostyles was significantly more than S-morphs and less than L-morph. On the anther, the ovule number per flower of homostyles was substantially less than both L-and S-morphs (p < .05; Table 2; Appendix S1). As a result, the P/O value TA B L E 2 Differences in floral characteristics and ancillary traits (mean ± SE) among different populations and morphs in Primula forbesii.

Floral traits Population
Mean

| Pollenviabilityandlongevity
The viabilities of L-and S-morph pollens reached their peaks on the second day after flowering (77.34 ± 1.68% and 78.67 ± 1.10%, respectively) and were significantly higher than homostyles (49.48

| Patternsofpollentubegrowthandselfincompatibility status
Our study showed that the pollen germinates on the stigma after 4 or 8 h of pollination in L-morphs and homostyles (Figure 4). We also found out that the pollen tubes arrive at the ovary in L-morphs after  (Figure 5f). A small number of irregular distorted and swollen pollen tubes also emerged but did not reach the ovary in intra-morph and self-pollination (Figure 5g,h).
The study revealed that the distylous plants in the intermorph pollination had the largest fruit set and seed production per fruit compared with all other pollination combinations ( Figure 6). The fruit sets of homostyles in intra-morph (93.56 ± 2.26%) and selfpollination (88.86 ± 2.83%) were significantly higher than those recorded in L-and S-morphs (p < .05) and close to distylous plants of was similar to those produced in L-morphs (p > .05) but significantly higher than those in S-morph (p < .05). In addition, they were all significantly lower than that of distylous plants of intermorph pollination (p < .05; Figure 6b). Under illegitimate pollination, the L-morphs showed partial fitness, with about 60% fruit set, while the S-morphs showed completely heteromorphic incompatibility, with no seed production.
The relative amount of terpenoids found in homostyles was significantly lower than in distylous flowers (p < .01), unlike the fatty acid derivatives, which were significantly higher in homostyles (p < .01; Table 3; Figure 7). The benzenoids did not differ considerably between homostyles and distylous flowers (p > .01). However, there were variations in some major floral scents (>1%) between homostyles and distylous individuals ( Table 3). For example, the amounts of linalool and alpha-terpineol were significantly higher in distylous flowers than in homostyles (p < .01). We also found substantially more cis-3-hexen-1-ol in homostyles than in distylous flowers (p < .01). We used the total peak area to represent floral scent intensity and found out that the concentration of floral volatile in S-morphs was significantly higher than in L-and H-morph flowers (p < .01).

| DISCUSS ION
Our analysis of the transitions from outcrossing to selfing in P.
forbesii obtained several key findings. We showed that the unreli-

| Ecologicalpressureofhomostylyevolution
Compared with the original habitats, the artificial cultivation or city environment may be characterized by increased spatial fragmentation and depauperate pollinator fauna, which would make the populations be affected by demographics, environment, and genetic stochasticity (Barmentlo et al., 2018;Oostermeijer et al., 2003).
When unreliable pollinators limit outcrossing seed sets, these unfavorable environments might favor variations in autonomous selfpollination for the production and spread of obligate outcrossing population, which is the Darwin's reproductive assurance hypothesis (Busch & Delph, 2012;Darwin, 1876;Moeller, 2006). The hypothesis is supported by the studies of numerous Primula species, in which self-incompatible distyly frequently breaks down into self-compatible homostyly (Carlson et al., 2008;Piper et al., 1986;Richards, 2003;Yuan et al., 2017). Based on higher herkogamy and the isoplethic equilibrium of L1:S1, outcrossing is more prevalent in wild populations of P. forbesii than the cultivated population. On the contrary, the cultivated population was biased to the partially selfcompatible L-morph and produced the completely self-compatible homostyly. We found that the main pollinators of the cultivated populations were the short-tongued A. cerana (about 5 mm), and the occasional visits by long-tongued bumblebees and butterflies have little contribution to this species. Because long-tongued pollinators mediate intermorph pollen transfer by contacting both the high-and low-level sex organs, they are considered more effective pollinators in distylous species with tubular flowers (Lloyd & Webb, 1992;Santos-Gally et al., 2013;Yuan et al., 2017). The corolla tube length of P. forbesii ranges from 5.43 to 6.67 mm, making it difficult for A. cerana to mediate disassortative pollen transfer between the anthers of L-morph and stigma of S-morph. Furthermore, the natural selection pressure due to the variable pollinator communities can rapidly alter the floral traits and mating system (Gervasi & Schiestl, 2017). Some variations in morphological and ancillary traits between wild and cultivated populations might be attributed to environmental conditions. For example, plants in the cultivated population had larger flowers with more nectar. The sufficient nutrient supply and stable growing condition of the artificial environment might be the main reasons for these differences, which are also supported by the significantly increased plant height and crown in the cultivated population (data not shown). In addition, the variation in pollen grain morphology among different populations is consistent with variations in previous studies (Li & Johnston, 2001;Xu et al., 2019). Morozowska and Idzikowska (2004) found significant differences in pollen morphologies between natural and cultivated populations in P. veris, which may be the result of adaptive change or/and random mutation due to geographical isolation.

| Originationofhomostyly
Homostyly originates from the rare crossovers or/and mutations of S-locus linkage genes governing distylous syndrome. In contrast F I G U R E 6 Differences in fruit set (a) and seed production per fruit (b) of Primula forbesii after controlled pollination in intermorph, intramorph and self-pollinated populations. *Significant differences at p < .05 or very significant at **p < .01; ***p < .001 based on the generalize linear model. Data are shown as means, and the bars indicate the standard errors.

(a) (b)
with the gradual evolution of self-pollination characteristic of most angiosperm groups, the variations of the mating system in distylous species could arise rapidly in a few generations (Kappel et al., 2017).
The long homostyly, which is the most common type, combines the female organ of L-morph with long papillae cells and the male organ of S-morph with large pollen grains and is usually associated with the breakdown of heteromorphic self-incompatibility and a high capability of autonomous self-fertilization (Barrett, 1992;Barrett & Shore, 2008;Haddadchi & Fatemi, 2015;Li & Johnston, 2001). The vast majority of homostyly in Primula species arose from this pathway (Ernst, 1955;Xu et al., 2019;Yuan et al., 2017;Zhou et al., 2017).
Recent studies of genetic architecture reveal that the S-linkage group governing Primula distylous syndrome is a hemizygous region of several genes that is only present in S-morph (S-) and absent in TA B L E 3 Major (>1%) floral scent compounds (mean ± SE) in three flower morphs of Primula forbesii. L-morph (--) (Li et al., 2016). This finding indicates that the long homostyly more likely originated from mutations of large effect genes in the S region rather than homologous recombination. Another alternative pathway of homostyly origin involved the modifier genes unlinked with S-locus. Through this approach, long homostyly might arise directly from the variations of the stamen of L-morph or pistil of S-morph, and it possesses pollen size, stigma papillae, and selfincompatibility similar to L-or S-morphs (Brys & Jacquemyn, 2015;Ganders, 1979;Mather & de Winton, 1941). Additionally, the long homostyly caused by polygenic modifiers would present strong variations of sexual organ height (Zhang et al., 2021).
Unfortunately, our data did not determine which evolutionary pathway the homostyly of P. forbesii originates from but supported more mutations of the S-locus linkage group. Based on similar stigma papillae and pollen size to L-and S-morph, respectively, long ho-

| Evolutionaryconsequencesofhomostyly
Comparisons of the ecologically functional characteristics between distyly and homostyly can reveal the consequences of pollination and reproductive biology on the variations of the mating system (Yuan et al., 2017). In our study, the flowers of P. forbesii   self-pollination (Barrett & Shore, 1987;de Vos et al., 2012;Ganders et al., 1985;Takebayashi et al., 2006;Wu et al., 2017;Yuan et al., 2017). Additionally, the transitions from distyly to homostyly in P. forbesii were also associated with reduced corolla diameter, similar to other Primula species (Li & Johnston, 2001;Zhong et al., 2019;Zhou et al., 2017). Since pollinators are no longer necessary, the floral traits associated with pollination can be reduced through relaxed selection or genetic drift, leading to selfing syndrome (Barrett et al., 2009;Goodwillie et al., 2010;Sicard & Lenhard, 2011). However, selfing syndrome is not inevi- In many Primula species, S-morph has stricter heteromorphic self-incompatibility than L-morph (Chen, 2009;Huang et al., 2015;Jiang & Li, 2017;Wedderburn & Richards, 1990). This is consistent with our results in which the L-morphs were partially selfcompatible, and S-morphs were completely self-incompatible.
Moreover, the illegitimate pollen tube on L-morph styles partially entered the ovary but could not germinate on the S-morph stigma. The performances of incompatible pollen indicate that the female incompatibility of S-morph is entirely sporophytic but is gametophytic in L-morph (Lewis & Jones, 1992;McCubbin, 2008).
Recently, Huu et al. (2022) demonstrated that concentrations of brassinosteroids during the development of female organs of P.
forbesii probably involve style elongation and the formation of female self-incompatibility, which provides new insights into heteromorphic self-incompatibility study. The high selfing rate of homostyly is widely reported in distylous groups (Barrett, 2019;Barrett & Shore, 2008;Darwin, 1877;Ganders, 1979;Ganders et al., 1985). Compared with distylous individuals of P. forbesii, the homostyles were completely self-compatible with 90% fruit set.
Although the seed production due to self-pollination of homostyly was significantly lower than intermorph pollination of distyly, self-fertilized homostyles were preferred in self-incompatible distylous population when unreliable short-tongued A. cerana pollinators limited the effectively intermorph pollen transfer between L-and S-morph.
The floral scent promotes reproductive success by attracting pollination insects (Majetic et al., 2009). Previous studies found that floral scent varied significantly among different Primula species, but not between the L-and S-morphs of each species (Gaskett et al., 2005;Zeng et al., 2022). However, similar to Johnson et al. (2019), we found significant differences in the intensity and composition of floral scent between L-and the S-morphs of P.
forbesii, with reduced floral scent emission and terpenoids content in L-morph. We speculate that the different self-incompatibility between L-and S-morph may be the main reason of floral scent divergence (Kariyat et al., 2021). The transitions of mating system commonly accompany variations in floral fragrance in many angiosperm lineages (Ferrari et al., 2006;Doubleday et al., 2013;Kariyat et al., 2021;Petrén et al., 2021;). Zeng et al. (2022) showed that the shift from distyly to homostyly was accompanied by reduced floral scent emission and changed floral scent profile in P. oreodoxa. This is consistent with our investigations where the homostyles of P. forbesii had reduced floral scent intensity and different floral scent profiles from distylous flowers, especially of S-morphs.
The changes in mating style mediated by pollinator groups maybe the main reason for the floral odour differences (Gervasi & Schiestl, 2017). Furthermore, the relative content of terpenoids, especially linalool, was significantly lower in homostylous than in distylous plants. The terpenoids elicit positive guiding behaviors in honey-and bumblebees and exist in most bee-pollinated angiosperms (Dobson, 2006;Dotterl & Vereecken, 2010;Laloi et al., 2001;Parachnowitsch et al., 2013).
In our study, A. cerana prefered distylous plants, especially Smorphs, compared with homostyles. The low pollinator activity in homostylous P. oreodoxa has been reported, although with no differences in elevation and size among populations (Yuan et al., 2017). Carr et al. (2014) demonstrated that the outcrossed progeny of Mimulus guttatus significantly had a higher visiting frequency than the selffertilized offspring, although they came from the same population.
F I G U R E 9 Mean (±SE) visitation frequencies of the pollinator insects on different floral morphs of Primula forbesii (a) and the main pollinator Apis cerana Fabricius (b). *Significant differences at p < .05 based on one-way ANOVA. Vertical bars indicate the standard errors.

(a) (b)
This could be due to pollinators strongly discriminating against inbreds (Carr et al., 2015;Ivey & Carr, 2005). A considerable P. forbesii homostyles come directly from self-pollination, leading to divergence in floral scent profiles and reduced visiting frequency in homostylous plants. Moreover, earlier studies showed that pollinators' preference for outcrossing plants still existed, although the corolla size and pollination reward had been controlled (Carr et al., 2014(Carr et al., , 2015Ivey & Carr, 2005). Therefore, pollinator-mediated natural selection on floral odor may be stronger than flower morphology or color selection in some species (Byers et al., 2014;Parachnowitsch et al., 2013). Collectively, the variations in the floral scent emission and composition during transitions to homostyly seem likely to be associated with reduced pollinator visiting, but further empirical investigations are necessary to determine the relationship between the floral scent variation and pollinator visiting in P. forbesii.

ACK N OWLED G EM ENTS
The authors wish to acknowledge Xi-Lin Wang, Xing Lin, and Min-Jie Jiang for their assistance during the pollination work and Su-Ping Gao and Xian-Ming Yang for valuable discussions on the evolution of distyly. This work was supported by the National Natural Science Foundation of China (32001356).

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATAAVA I L A B I L I T YS TAT E M E N T
All data needed to evaluate the conclusions in the paper are present in the paper and/ or in the Supporting Information.